Elsevier

Neuroscience

Volume 147, Issue 1, 15 June 2007, Pages 60-70
Neuroscience

Cellular neuroscience
Beta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons

https://doi.org/10.1016/j.neuroscience.2007.03.047Get rights and content

Abstract

Neuronal death leading to gross brain atrophy is commonly seen in Alzheimer’s disease (AD) patients. Yet, it is becoming increasingly apparent that the pathogenesis of AD involves early and more discrete synaptic changes in affected brain areas. However, the molecular mechanisms that underlie such synaptic dysfunction remain largely unknown. Recently, we have identified dynamin 1, a protein that plays a critical role in synaptic vesicle endocytosis, and hence, in the signaling properties of the synapse, as a potential molecular determinant of such dysfunction in AD. In the present study, we analyzed beta-amyloid (Aβ)-induced changes in synaptic vesicle recycling in rat cultured hippocampal neurons. Our results showed that Aβ, the main component of senile plaques, caused ultrastructural changes indicative of impaired synaptic vesicle endocytosis in cultured hippocampal neurons that have been stimulated by depolarization with high potassium. In addition, Aβ led to the accumulation of amphiphysin in membrane fractions from stimulated hippocampal neurons. Moreover, experiments using FM1-43 showed reduced dye uptake in stimulated hippocampal neurons treated with Aβ when compared with untreated stimulated controls. Similar results were obtained using a dynamin 1 inhibitory peptide suggesting that dynamin 1 depletion caused deficiency in synaptic vesicle recycling not only in Drosophila but also in mammalian neurons. Collectively, these results showed that Aβ caused a disruption of synaptic vesicle endocytosis in cultured hippocampal neurons. Furthermore, we provided evidence suggesting that Aβ-induced dynamin 1 depletion might play an important role in this process.

Section snippets

Preparation of hippocampal cultures

Embryonic day (E) 18 rat embryos were used to prepare primary hippocampal cultures as previously described (Goslin and Banker, 1991). Briefly, hippocampi were dissected and freed of meninges. The cells were dissociated by trypsinization followed by trituration with a fire-polished Pasteur pipette. For electron microscopy and subcellular fractionation experiments, hippocampal neurons were plated at high density (500,000 cells/60-mm dish) in MEM with 10% horse serum (MEM10). After 2 h, the medium

Aβ caused accumulation of amphiphysin at the membrane of stimulated cultured hippocampal neurons

We have previously shown that Aβ induced dynamin 1 depletion in cultured hippocampal neurons. An expected consequence of dynamin 1 depletion is the disruption of synaptic vesicle endocytosis during synaptic activity. To obtain evidence of such Aβ potential effects, we used Western blot analysis to detect membranous accumulations of proteins involved in synaptic vesicle endocytosis in control hippocampal neurons and in neurons stimulated in the presence of high potassium stimulation for 10 min.

Discussion

The results presented herein indicated that Aβ impaired synaptic vesicle endocytosis, and hence the normal recycling of these organelles, during sustained synaptic activity in cultured hippocampal neurons. In addition, our data provided further evidence suggesting that these Aβ effects could be mediated, at least in part, by the significant depletion of dynamin 1 induced by this peptide. Together, these findings identify potential mechanisms underlying synaptic dysfunction in early stages of AD.

Acknowledgments

This study was supported by grant NIH/NS39080 to A.F. B.L.K was supported in part by NIA/AG20506 training grant and an American Foundation for Aging Research Fellowship. We thank Rachel Bergstrom and Roxanna Sinjoanu for excellent technical support. We also thank Linda Juarez (RRC EM facility at University of Illinois–Chicago) for her technical support with the electron microscopy.

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